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. 2001 Sep 15;21(18):7194-202.
doi: 10.1523/JNEUROSCI.21-18-07194.2001.

Insulin-like growth factor-I is necessary for neural stem cell proliferation and demonstrates distinct actions of epidermal growth factor and fibroblast growth factor-2

Y Arsenijevic  1 , S Weiss, B Schneider, P Aebischer
Affiliations

Insulin-like growth factor-I is necessary for neural stem cell proliferation and demonstrates distinct actions of epidermal growth factor and fibroblast growth factor-2

Y Arsenijevic et al. J Neurosci. .

Abstract

Neural stem cells (NSCs), when stimulated with epidermal growth factor (EGF) or fibroblast growth factor-2 (FGF-2), have the capacity to renew, expand, and produce precursors for neurons, astrocytes, and oligodendrocytes. We postulated that the early appearance of insulin-like growth factor (IGF-I) receptors during mouse striatum development implies a role in NSC regulation. Thus, we tested in vitro the action of IGF-I on the proliferation of striatal NSCs. In the absence of IGF-I, neither EGF nor FGF-2 was able to induce the proliferation of E14 mouse striatal cells. However, addition of IGF-I generated large proliferative clusters, termed spheres, in a dose-dependent manner. The newly generated spheres were multipotent, and clonal analysis revealed that EGF or FGF-2, in the presence of IGF-I, acted directly on NSCs. The actions of IGF-I suggest distinct modes of action of EGF or FGF-2 on NSCs. First, continuous versus delayed administration of these neurotrophic factors showed that neither IGF-I nor EGF had an effect on NSC survival, whereas FGF-2 promoted the survival or maintenance of the stem cell state of 50% of NSCs for 6 d. Second, short-term exposure to IGF-I induced the proliferation of NSCs in the presence of EGF, but not of FGF-2, through an autocrine secretion of IGF-I. These findings suggest that IGF-I is a key factor in the regulation of NSC activation and that EGF and FGF-2 control striatal NSC proliferation, in part, through distinct intracellular mechanisms.

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Figures

Fig. 1.
Fig. 1.
Generation of striatal spheres requires IGF-I. Primary striatal E14 cells were plated at 5000 cells per well in a 96-well plate in the presence of EGF (A) or FGF-2 (B) and increasing concentrations of IGF-I. Eight to 10 d later, the proliferating cells generating “spheres” were counted. Six to eight wells were analyzed per condition. The graphs show a representative dose–response curve of three wells for each group. Note that in the absence of IGF-I, no spheres were formed.
Fig. 2.
Fig. 2.
FGF-2 + IGF-I and EGF + IGF-I generated spheres are multipotent. Single spheres were individually transferred to culture wells and exposed to conditions that favor cell differentiation (see Materials and Methods). The majority of the analyzed spheres that were generated by either FGF-2 (A–C) or EGF (D–F) (Table 1) contained cells immunoreactive for the neuronal marker β-tubulin (A,D, arrows), the astrocyte marker GFAP (B, arrowhead), and the oligodendrocyte marker O4 (F, arrowhead). Magnification, 400×.
Fig. 3.
Fig. 3.
Clonal analysis reveals a direct action of IGF-I on NSCs. The effect of EGF and IGF-I was tested with striatal E14 cells that were plated at clonal density (30–150 cells per 96-well plate). Cells were followed continuously. Between 2 and 5 DIV, rare cells enlarged (A) and divided (B) to form a sphere (C) after 10–15 DIV. Similar observations were made for cells responsive to IGF-I + FGF-2 (data not shown) (Table 2). Magnification, 400×.
Fig. 4.
Fig. 4.
FGF-2 maintains in vitro the presence of embryonic striatal NSCs. A, To test the possible survival effect of EGF or IGF-I on primary E14 striatal cells, one factor was present during the whole experimental period, whereas the other was administered 3–4 or 5–6 d after plating, and the spheres were counted 8–10 DIV after the final addition. For cell density conditions, see the legend to Figure 1. The co-incubation of EGF + IGF-I beginning at plating served as control. B, All delayed administrations of EGF or IGF-I resulted in a significant decrease in sphere number in comparison to the control group (B; n = 5). C, The constant presence of FGF-2 sustained NSC survival or the NSC state for at least 6 DIV (B; n = 3–5). *p < 0.05, **p < 0.01, ***p < 0.001, #p = 0.2, in comparison to the control group.
Fig. 5.
Fig. 5.
EGF and FGF-2 are both acting on the same stem cells. A, To know whether IGF-I, EGF, and FGF-2 are acting on the same cells in our culture conditions (5000 cells per well), the additive action of these factors was tested. We have arbitrarily chosen IGF-I + FGF-2-generated sphere number as the reference group. No significant differences were observed between the groups. Note that no additive action of IGF-I, EGF, and FGF-2 was observed (n = 5). B, To confirm these results, the action of FGF-2 on survival or the maintenance of the NSC state was assayed on EGF-responsive stem cells. Substitution of FGF-2 by EGF + IGF-I at 4 DIV gave rise to a similar number of spheres in comparison to cells stimulated by FGF-2 and IGF-I constantly (second column) or starting at 4 DIV (third column). Note that FGF-2 acts as a survival factor or maintains NSC state for both FGF-2- and EGF-responsive stem cells. C, Control culture without factors.
Fig. 6.
Fig. 6.
Short exposure to IGF-I is sufficient to induce NSC proliferation in the presence of EGF, but not of FGF-2. Striatal cells from mouse E14 embryos were stimulated with EGF for up to 10 DIV. During EGF treatment, groups of cells received various time exposures of IGF-I: 10 DIV, 24 hr, or 2 hr. Only cells that were stimulated with IGF-I induced sphere formation. No significant differences in sphere numbers were observed between long- and short-term (24 and 2 hr) stimulation with IGF-I (second and third columns, p = 0.78). Short exposure of IGF-I during the constant presence of FGF-2 rarely generated spheres (n = 5; **p < 0.01).
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